C
Synlett
X. Li et al.
Letter
The fragmentation of the d -VX adducted nonapeptide
Figure 2 and Figure 3) was observed by precursor-to-prod-
(2) (a) Morita, H.; Yanagisawa, N.; Nakajima, T.; Shimizu, M.;
Hirabayashi, H.; Okudera, H.; Nohara, M.; Midorikawa, Y.;
Mimura, S. Lancet 1995, 346, 290. (b) Nakajima, T.; Ohta, S.;
Morita, H.; Midorikawa, Y.; Mimura, S.; Yanagisawa, N. J. Epide-
miol. 1998, 8, 33. (c) Suzuki, T.; Morita, H.; Ono, K.; Maekawa,
K.; Nagai, R.; Yazaki, Y. Lancet 1995, 345, 980.
5
(
uct ion transitions monitored using multiple reaction mon-
itoring (MRM). The protonated peptide-adduct fragments
showed losses of the adducted agent, the C-terminal serine
and the C-terminal alanine. It showed the same fragmenta-
(
3) (a) Shih, M. L.; Smith, J. R.; McMonagle, J. D.; Dolzine, T. W.;
Gresham, V. C. Biol. Mass Spectrom. 1991, 20, 717. (b) Black, R.
M.; Clarke, R. J.; Read, R. W.; Reid, M. T. J. J. Chromatogr. A 1994,
7c
tion pattern as the VX-BuChE nonapeptide. As shown in
the Figure 3, the precursor ion for the chemical 7 (m/z =
6
1
7
62, 301. (c) Tørnes, J. A. Rapid. Commun. Mass Spectrom. 1996,
0, 878. (d) Black, R. M.; Read, R. W. J. Chromatogr. A 1997, 759,
9. (e) Black, R. M.; Read, R. W. J. Chromatogr. A 1998, 794, 233.
9
07.3968) exhibited product ions derived from a loss of
129.0604 Da which corresponds to the loss of the d -VX
5
moiety (m/z = 778.3364); further losses of the terminal ser-
ine (m/z = 673.2932), and the alanine (m/z = 602.2554) resi-
due were also exhibited.
(f) Noort, D.; Hulst, A. G.; Platenburg, D. H. J. M.; Polhuijs, M.;
Benschop, H. P. Arch. Toxicol. 1998, 72, 671.
(
4) Riches, J.; Morton, I.; Read, R. W.; Black, R. M. J. Chromatogr. B
2
005, 816, 251.
5) (a) Bao, Y.; Liu, Q.; Chen, J.; Lin, Y.; Wu, B.; Xie, J. J. Chromatogr. A
012, 1229, 164. (b) Noort, D.; Benschop, H. P.; Black, R. M. Toxi-
(
2
col. Appl. Pharmacol. 2002, 184, 116.
(6) Abney Carter, W.; Knaack, L. S. J.; Ali, I. A. A.; Johnson, C. R.
Chem. Res. Toxicol. 2013, 26, 775.
(
7) (a) Knaack, S. J.; Zhou, Y.; Abney, W. C.; Jacob, T. J.; Prezioso, M.
S.; Hardy, K.; Lemire, W. S.; Thomas, J.; Johnson, C. R. Anal.
Chem. 2012, 84, 9470. (b) Carter, D. M.; Crow, S. B.; Pantazides,
G. B.; Watson, M. C.; Thomas, D. J.; Blake, T. A.; Johnson, C. R.
Anal. Chem. 2013, 85, 11106. (c) Sporty, L. S. J.; Lemire, W. S.;
Jakubowski, M. E.; Renner, A. J.; Evans, A. R.; Williams, F. R.;
Schmidt, G. J.; van der Schans, J. M.; Noort, D.; Johnson, C. R.
Anal. Chem. 2010, 82, 6593.
Figure 3 Product ion scan of protonated d -VX –adducted nonapep-
5
tide
(
8) (a) He, Z.-J.; Wang, Y.-M.; Tang, C.-C. Phosphorus, Sulfur Silicon
Relat. Elem. 1997, 127, 59. (b) Bennet, A. J.; Kovach, I.; Bibbs, J. A.
J. Am. Chem. Soc. 1989, 111, 6224.
In summary, a synthesis approach of d -VX adducted
5
nonapeptide via solid phase has been developed.11 The
(
9) (a) Hudson, R. F.; Keay, L. J. Chem. Soc. 1956, 2463. (b) Struck, R. F.
MS/MS fracture manner of the d -VX nonapeptide is the
5
7c
J. Med. Chem. 1966, 9, 231.
same as that of VX-BuChE nonapeptide. So the brand new
(
(
10) MacDonald, M.; Lanier, M.; Cashman, J. Synlett 2010, 1951.
11) The target compound d -VX adducted nonapeptide was synthe-
compound d -VX peptide could be used as the isotope-la-
5
5
beled internal standard for LC–MS/MS-detecting BuChE-
sized via the solid-phase peptide synthesis manually from C-
terminal to N-terminal to enhance the coupling efficiency. The
peptides were synthesized using Fmoc chemistry on a Fmoc-Ser
(t-Bu)-Wang resin and HBTU/DIPEA activation. Piperidine–DMF
(25%) was used to deprotect the Fmoc group. The rest of the
amino acid residues were induced onto the resin in sequence by
HBTU/DIPEA activation. The Kaiser test was used to ensure the
connection with right amino acid residue upon every step. After
this point, cleavage from the resin was achieved by treating the
peptide resin with the solution containing 68.5% TFA, 10% 1,2-
ethanedithiol, 10% thioanisole, 5% phenol, 3.5% double-distilled
water, and 1% triisopropylsilane for 3 h at r.t. The crude product
of the phosphorylated peptide which could be precipitated by
OPNA adducts. Additionally, the synthesis material d -Etha-
6
nol used in the procedure has a much more easier availabil-
ity and cheaper price than the deuterated amino acid used
5
in the former literature. This method may also find appli-
cations in the synthesis and detection of other phosphory-
lated nonapeptides.
Acknowledgment
The work was supported by the State Key Laboratory of NBC Protec-
tion for Civilian (grant no. SKLNBC2014-06).
anhydrous Et O was further purified by reversed-phase HPLC
2
on a C18 column (0.05% TFA–water–2% MeCN). Peptide purity
was>90% by analysis of HPLC-DAD. It was also verified by NMR
Supporting Information
and high-resolution MS. HRMS (ESI+): m/z found for
1
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0036-1588691.
C
36
H
51
D
5
PN
9
O
16: 907.3968. H NMR (599.7 MHz, D
2
O): δ =1.28
2
S
u
p
p
ortioIgnfrm oaitn
S
u
p
p
ortioIgnfrm oaitn
(t, J = 7.4 Hz, 9 H), 1.47 (d, JH–C–P = 17.6 Hz, 3 H), 1.80–2.11 (m, 6
H), 2.37 (t, J = 7.1 Hz, 2 H), 3.12 (m, 3 H), 3.75–3.88 (m, 6 H),
4
.20–4.35 (m, 9 H), 4.55 (s, 1 H), 7.18 (d, J = 7.2 Hz, 2 H), 7.26–
13
1
References
7.31 (m, 3 H). C{ H} NMR (150.8 MHz, D
74.7, 174.6, 173.5, 173.3, 170.9, 170.7, 169.8, 169.6, 133.6,
2
O): δ = 177.1, 175.0,
1
(
1) (a) John, H.; Worek, F. Anal. Bioanal. Chem. 2008, 391, 97.
129.3, 129.1, 128.0, 64.2, 64.1, 61.2, 61.1, 55.1, 54.4, 53.45,
53.40, 53.35, 53.1, 49.9, 49.5, 49.4, 42.2, 36.7, 30.0, 26.1, 16.7,
(b) Fidder, A.; Hulst, A. G.; Noort, D.; de Ruiter, R.; van der
Schans, M. J.; Benschop, H. P.; Langenberg, J. P. Chem. Res. Toxi-
col. 2002, 15, 582.
16,4, 9.4, 8.5. 31P{
H} NMR (242.8 MHz, D O): δ = 35.9.
2
1
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–C